PT86643B - METHOD OF MEASURING ANTI-DNA ANTIBODIES - Google Patents
METHOD OF MEASURING ANTI-DNA ANTIBODIES Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Os soros de pacientes que sofrem de lupus eritematoso sistémico (SLE) contêm auto-anticorpos que podem reagir com uma variedade de componentes nucleares incluindo ácidos nucleicos, proteínas nucleares e várias combinações de DNA de cadeia dupla(dsDNA) e de DNA de cadeia simples (ssDNA). Alguns reagem com bases ou I nucleotídeos individuais, com guanina e com sequências oligo (dt) e mesmo com polinucleotideos sintéticos. Schwartz, R.S. e Stollar, D.J. Clin. Invest. 75:321(1985). Isenberg, D.A., Madaio, M.P., Reichlin, M., Schoenfeld, Y., Ranch, J.,Stollar, D. e Schwartz, R.S., Lancet, 25 de Agosto ) 1984. Ainda, estes auto-anticorpos reagem com antigénios do tipo lípido complexo como sejam cardiolipina e outros fosfolípidos das membranas de plaquetas, antipoli (ADP-ribo se), histonas e com proteoglicanos; nomeadamente, ácido bria lurónico e sulfato de condroitina. No entanto, a reacção de anticorpos com dsDNA ou DNA nativo (nDNA) é a principal marca serológica desta doença e a capacidade para medir dsDNA mostrou-se particularmente útil no diagnóstico e controle da actividade da doença.Sera from patients suffering from systemic lupus erythematosus (SLE) contain autoantibodies that can react with a variety of nuclear components including nucleic acids, nuclear proteins and various combinations of double-stranded DNA (dsDNA) and single-stranded DNA ( ssDNA). Some react with individual bases or nucleotides, with guanine and with oligo (dt) sequences and even with synthetic polynucleotides. Schwartz, R.S. and Stollar, D.J. Clin. Invest. 75: 321 (1985). Isenberg, DA, Madaio, MP, Reichlin, M., Schoenfeld, Y., Ranch, J., Stollar, D. and Schwartz, RS, Lancet, August 25) 1984. In addition, these autoantibodies react with antigens from the complex lipid type such as cardiolipin and other platelet membrane phospholipids, antipoly (ADP-ribo se), histones and with proteoglycans; namely, luronic acid and chondroitin sulfate. However, the reaction of antibodies with dsDNA or native DNA (nDNA) is the main serological feature of this disease and the ability to measure dsDNA has proved to be particularly useful in the diagnosis and control of disease activity.
Têm sido idealizados muitos métodos de ensaio para detectar e semi-quantificar dsDNA | em pacientes com SLE. Estes métodos usados para identificar estes auto-anticorpos têm incluído difusão em gel, Seligmann, M., C.R.Acad.Sci (Paris) , 245:243 (1957); fixação do complemento; Robbins, W.C., Holman, H.R., Deicher, H. e kunkel, H.G., Proc. Soc. Exp.Biol. (NY), 96:575 (1957); I anafilaxia cutânea passiva; Deicher, H.R.G. Holmann, H.R. ,Many test methods have been devised to detect and semi-quantify dsDNA | in SLE patients. These methods used to identify these autoantibodies have included gel diffusion, Seligmann, M., C.R.Acad.Sci (Paris), 245: 243 (1957); complement fixation; Robbins, W.C., Holman, H.R., Deicher, H. and kunkel, H.G., Proc. Soc. Exp.Biol. (NY), 96: 575 (1957); I passive skin anaphylaxis; Deicher, H.R.G. Holmann, H.R.,
Kunkel, H.G. e Ovarg, Z. , J.Immunol 84:106(1960); aglutinação com bentonite,; Bozicavich, J. Nason, J.P. e Kayhoe,Kunkel, H.G. and Ovarg, Z., J. Immunol 84: 106 (1960); agglutination with bentonite; Bozicavich, J. Nason, J.P. and Kayhoe,
D.E., ProcSoc.Exp.BIol.(N.Y.), 103:636 (1960); hemaglutinação; Jokinen, E.J. e Julkunen, H., Ann. Rlseum.Dis. 24:4 77 (1965); radioelectroforese; Bickel, Y.B., Barnett, E.V. e Pearson C. M. Clin. Exp.Immunol 3:641(1968);radioensaiosD.E., ProcSoc.Exp.BIol. (N.Y.), 103: 636 (1960); hemagglutination; Jokinen, E.J. and Julkunen, H., Ann. Rlseum.Dis. 24: 47 (1965); radioelectrophoresis; Bickel, Y.B., Barnett, E.V. and Pearson C. M. Clin. Exp.Immunol 3: 641 (1968); radioassays
125 usando H ou I; Wold, R.T., Yonng, F.E., Tan, E.M.125 using H or I; Wold, R.T., Yonng, F.E., Tan, E.M.
e Farr, R.S. Science 161:806 (1968); e imunoensaios comand Farr, R.S. Science 161: 806 (1968); and immunoassays with
enzimas; Rubin, R.L. Joslin, F.G. e Tan, E.M. J.Immunol. Methods 63:359 (1983). A maior parte, senão todos, os métodos atrás referidos carecem de especificidade e de sensibilidade. Em muitos casos são relatados falsos positivos devido à falta de especificidade diagnóstico que não permite diferenciar SLE de outras doenças do tecido conjuntivo.enzymes; Rubin, R.L. Joslin, F.G. and Tan, E.M. J. Immunol. Methods 63: 359 (1983). Most, if not all, of the aforementioned methods lack specificity and sensitivity. In many cases, false positives are reported due to the lack of diagnostic specificity that does not allow SLE to be differentiated from other connective tissue diseases.
Verificou-se que os radioensaios e imunoensaios com enzimas têm dado uma correlação aceitável com o grau e gravidade dos estados de SLE. No entanto, estes métodos imunológicos isam todos preparações marcadas de nDNA derivado do timo de vitela que está altamente polimerizado com fragmentos de DNA de peso molecular elevado e comprimento variável, em média cerca de 20 quilopares de bases. Estas preparações contêm igualmente quantidades variáveis de ssDNA.It was found that radioassays and enzyme immunoassays have given an acceptable correlation with the degree and severity of SLE states. However, these immunological methods are all labeled preparations of nDNA derived from the calf's thymus which is highly polymerized with DNA fragments of high molecular weight and variable length, on average about 20 kilopares of bases. These preparations also contain varying amounts of ssDNA.
Estas moléculas de DNA de 20 kpb muitas vezes ligam-se inespecificamente a proteinas circulantes do plasma, enquanto que o ssDNA contaminante nestas preparações se liga ao Csq do complemento.These 20 kbp DNA molecules often bind non-specifically to circulating plasma proteins, while the contaminating ssDNA in these preparations binds to the complement Csq.
Assim, estes métodos imunológicos convencionais para medição de anticorpos contra dsDNA (nDNA) geralmente requerem pré-tratamento da amostra de soro ou de plasma a 56C durante 30 minutos para inactivar o Csq do complemento antes da reacção imunológica entre o DNA traçador e os anticorpos anti-DNA circulantes.Thus, these conventional immunological methods for measuring antibodies to dsDNA (nDNA) generally require pretreatment of the serum or plasma sample at 56C for 30 minutes to inactivate the complement Csq before the immunological reaction between the tracer DNA and the anti antibodies -DNA circulating.
Nas condições ideais, preferencialmente uma molécula de IgG circulante anti-DNA liga-se apenas a uma ou a duas moléculas de dsDNA marcado de um dado comprimento e sem contaminantes, particularmente ssDNA.Ideally, preferably a circulating anti-DNA IgG molecule binds only to one or two molecules of labeled dsDNA of a given length and free of contaminants, particularly ssDNA.
Com esta finalidade foram projectadas técnicas in vivo para preparar dsDNA marcado(esi h r Ί O C pecialmente marcado comlz:3i) através da introdução de I5-For this purpose, in vivo techniques were designed to prepare labeled dsDNA (esi hr Ί OC specially labeled with lz: 3 i) by introducing I5-
-desoxicitidina em células HeLa. No entanto, estes métodos de marcação in vivo não produzem dsDNA marcado com um grau de pureza elevado devido aos contaminantes de ssDNA normalmente encontrados em tais preparações como sub-produtos.-deoxycytidine in HeLa cells. However, these in vivo labeling methods do not produce high purity labeled dsDNA due to the ssDNA contaminants normally found in such preparations as by-products.
De acordo com este invento, os anticorpos circulantes anti-DNA presentes em fluidos biológicos são medidos, por exemplo, por radioensaio usando um traçador bem definido de I-ds DNA com um determinado número de quilopares de bases, preparado por técnicas de DNArecombinante e sem quaisquer contaminantes de ssDNA. 0 plasmídeo para a preparação in vitro de tal traçador, parâmetros de ensaios e o kit para medição dos anticorpos anti-DNA são aqui descritos seguidamente.According to this invention, circulating anti-DNA antibodies present in biological fluids are measured, for example, by radioassay using a well-defined tracer of I-ds DNA with a certain number of base kilopares, prepared by DNArecombinant techniques and without any ssDNA contaminants. The plasmid for the in vitro preparation of such a tracer, assay parameters and the kit for measuring anti-DNA antibodies are described hereinafter.
((
Sumário do inventoSummary of the invention
Resumidamente o presente invento compreende um método para medição de títulos de anticorpos anti-DNA em fluidos biológicos por um método imunológico usando uma marca de dsDNA sem fragmentos de ssDNA, em que a marca de dsDNA é preparada por técnicas de DNA recomI binante, tendo um comprimento constante de kpb bem definidas | e uma determinada actividade específica, e tal do DNA marcado sendo usado como traçador no referido método imunológico.Briefly the present invention comprises a method for measuring anti-DNA antibody titers in biological fluids by an immunological method using a dsDNA tag without ssDNA fragments, wherein the dsDNA tag is prepared by recombinant DNA techniques, having a constant length of well-defined kpb | and a specific activity, such as the labeled DNA being used as a tracer in said immunological method.
I invento também compreende um kit de ensaios para medição de títulos de anticorpos anti-DNA em fluidos biológicos por um método imunológico usando uma marca de dsDNA sem fragmentos de ssDNA, em que a marca de dsDNA é preparada por técnicas de DNA recombinante, tendo um comprimento constante de kpb bem definidas e uma determinada actividade específica, e tal dsDNA marcado sendo usado como traçador no referido método imunológico.The invention also comprises an assay kit for measuring anti-DNA antibody titers in biological fluids by an immunological method using a dsDNA tag without ssDNA fragments, in which the dsDNA tag is prepared by recombinant DNA techniques, having a constant length of well-defined kpb and a specific activity, and such labeled dsDNA being used as a tracer in said immunological method.
Constitui um objectivo do presente invento ultrapassar as desvantagens referidas atrás nos métodos imunológicos e em particular radioensaios com dsDNA 125 marcado em I e outros ensaios com dsDNA marcado.It is an objective of the present invention to overcome the disadvantages mentioned above in immunological methods and in particular radioassays with I-labeled dsDNA 125 and other assays with labeled dsDNA.
Em geral, constitui um objectivo deste invento, proporcionar um novo método para medição de títulos anti-DNA em fluidos biológicos.In general, it is an object of this invention to provide a new method for measuring anti-DNA titers in biological fluids.
É também um objectivo geral deste invento proporcionar um novo kit de ensaio para efectuar as medições em fluidos biológicos préviamente discutidos.It is also a general objective of this invention to provide a new test kit for making measurements on biological fluids previously discussed.
Estes e outros objectivos e vantagens deste invento serão aparentes a partir da discusão mais detalhada que se segue, acompanhada dos desenhos juntos.These and other objectives and advantages of this invention will be apparent from the more detailed discussion that follows, accompanied by the accompanying drawings.
-ΊDescrição detalhada do invento objectivo do presente inven- i to é projectar uma molécula de dsDNA de pares de bases bem definidos, com um determinado comprimento e de modo a que ; tal preparação não tenha ssDNA contaminante. Tal prepara- J ção quando marcada com uma sonda produtora de um sinal (iodo 125, marca enzimática, marca fluorescente ou marca quimio- , luminescente ou bioluminescente) terá uma actividade específica bem definida, será estável e ligar-se-á especificamente a anticorpos circulantes anti-DNA em soro ou plasma SLE.- Detailed description of the invention The objective of the present invention is to design a well-defined base pair dsDNA molecule, with a certain length and so that ; such preparation has no contaminating ssDNA. Such preparation when labeled with a probe producing a signal (iodine 125, enzyme label, fluorescent label or chemo-, luminescent or bioluminescent label) will have a well-defined specific activity, be stable and will specifically bind to antibodies circulating anti-DNA in serum or SLE plasma.
Para se conseguir tal objectivo construiu-se um DNA de plasmídeo que foi preparado a partir de Escherichia coli sem conter ssDNA. Este plasmídeo foi cortado com uma enzima de restrição adequada para pro- l i duzir dsDNA de um determinado comprimento que não excedaTo achieve this goal, a plasmid DNA was constructed that was prepared from Escherichia coli without containing ssDNA. This plasmid was cut with an appropriate restriction enzyme to produce dsDNA of a certain length that does not exceed
1,5 kpb a ser usado como material traçador. 0 presente plasmídeo usado no presente invento divulgado é preparado a partir de E.coli sendo no entanto possível usar qualquer outro organismos adequados contendo plasmideos, tais como Bacillus subtilis, Pseudomonas putida e Saccharomyces cerevisial. Outros organismos contendo plasmideos serão obvios para os familiarizados com a matéria, Ainda, dsDNA obtido por sintese química pode também ser usado para atingir os objectivos do presente invento.1.5 kbp to be used as tracing material. The present plasmid used in the present disclosed invention is prepared from E.coli however it is possible to use any other suitable organisms containing plasmids, such as Bacillus subtilis, Pseudomonas putida and Saccharomyces cerevisial. Other plasmid-containing organisms will be obvious to those of ordinary skill in the art. Furthermore, dsDNA obtained by chemical synthesis can also be used to achieve the objectives of the present invention.
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Este invento será descrito detalhadamente com referência aos exemplos que se seguem os quais são apresentados apenas com fins ilustrativos.This invention will be described in detail with reference to the following examples which are presented for illustrative purposes only.
Exemplo 1Example 1
Preparação de DNA de plasmídeo, pNDPC 1Preparation of plasmid DNA, pNDPC 1
A Figura 1 mostra um mapa de enzimas de restrição do DNA de plasmídeo, pNDPC 1 que é usado nos exemplos deste invento.Figure 1 shows a map of plasmid DNA restriction enzymes, pNDPC 1 which is used in the examples of this invention.
plasmideo pNDPC 1 foi constriudo como se segue: pUC18 (Yarnisch-Perron, C. et;al, Gene 33:103(1985) foi parcialmente digerido com Cfr I ou Ban I e depois autoligado por DNA ligase de T4 como se mostra na Figura 1. As marcas selectivas foram Ap e lac. pNDPC com 2431 pares de bases foi inserido em E.coli K12 Jm 109 por processos convencionais e a bactéria cultivada num meio LB (1% de triptona, 0,5% de extracto de levedura, 2,5% NaCl). O plasmídeo foi então purificado por processos regulares.plasmid pNDPC 1 was constructed as follows: pUC18 (Yarnisch-Perron, C. et; al, Gene 33: 103 (1985) was partially digested with Cfr I or Ban I and then self-ligated by T4 DNA ligase as shown in Figure 1. The selective marks were Ap and lac.pNDPC with 2431 base pairs was inserted into E.coli K12 Jm 109 by conventional processes and the bacteria cultivated in an LB medium (1% tryptone, 0.5% yeast extract, 2.5% NaCl) The plasmid was then purified by regular procedures.
O pNDPCl foi então cortado com a enzima de restrição Cfr I ou Bam I numa solução aquosa contendo tampão 10mM Tris-HCl pH8,5 e contendo 7mM MgCl2, 20mM NaCl e 7mM 2-mercaptoetanol. O dsDNA obtido usado Cfr I continha fragmentos de 0,9 e 1,4 kpb enquanto que o dsDNA obtido usando Ban I continha fragmentos de 1,1 e 1,2 kpb.The pNDPCl was then cut with the restriction enzyme Cfr I or Bam I in an aqueous solution containing 10mM Tris-HCl pH8.5 buffer and containing 7mM MgCl 2 , 20mM NaCl and 7mM 2-mercaptoethanol. The dsDNA obtained using Cfr I contained fragments of 0.9 and 1.4 kbp while the dsDNA obtained using Ban I contained fragments of 1.1 and 1.2 kbp.
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II
Exemplo 2Example 2
Iodinação do dsDNA do Exemplo 1Iodination of the dsDNA of Example 1
Os métodos de iodinação in vitro para marcação de moléculas de DNA recombinante incluem nick-translation, polinucleotídeo cinase, DNA polimerase fragmento grande (enzima klenow), DNA polimerase de T4 e desoxitransferase terminal (TdT).In vitro iodination methods for labeling recombinant DNA molecules include nick-translation, polynucleotide kinase, large fragment DNA polymerase (klenow enzyme), T4 DNA polymerase and terminal deoxytransferase (TdT).
II
I método de nick-translation I i dá a actividade específica mais elevada mas algum do dsDNA marcado parece dar origem a ssDNA e portanto é inadequado para os presentes fins. Por outro lado, os métodos de iodinação usando polinucleotídeo cinase, enzima klenow, DNA polimerase de T4 ou TdT em que os terminais do DNA são marcados, produzem traçadores de actividade moderada comparado com o método de nick-translation, mas não originam fragmentos de ssDNA. Estes métodos de iodinação ou marcação são portanto os preferidos para este invento mas não devem ser considerados restrictivos. Outros métodos de marcação serão obvios para os familiarizados com a matéria.The nick-translation method I i gives the highest specific activity but some of the labeled dsDNA appears to give rise to ssDNA and is therefore unsuitable for the present purposes. On the other hand, iodination methods using polynucleotide kinase, klenow enzyme, T4 or TdT DNA polymerase in which the DNA terminals are labeled, produce tracers of moderate activity compared to the nick-translation method, but do not originate ssDNA fragments . These methods of iodination or labeling are therefore preferred for this invention but should not be considered restrictive. Other marking methods will be obvious to those familiar with the subject.
O dsDNA (1,1-1,2 kpb) obtido no Exemplo 1, supra, foi dissolvido numa solução aquosa contendo tampão 50 m.K Tris-HCl, pH 7,2 e contendo MgSO^, 1 mM } ditiotreitol, 500 ug/ml de albumina de soro bovina. Adicio-The dsDNA (1.1-1.2 kbp) obtained in Example 1, supra, was dissolved in an aqueous solution containing 50 mK Tris-HCl buffer, pH 7.2 and containing MgSO4, 1 mM} dithiothreitol, 500 µg / ml of bovine serum albumin. Add-
25 nou-se dGTP, dATP e dTTP, lmM de cada e 1 mCi de I-25 dGTP, dATP and dTTP, 1 mM of each and 1 mCi of I-
-dCTP e 25 unidades de DNA polimerase de T4 , fragmento gran de (enzima Kelenow) e deixou-se decorrer a reacção, à temperatura ambiente durante 30 minutos.-dCTP and 25 units of T4 DNA polymerase, large fragment (Kelenow enzyme) and the reaction was allowed to proceed at room temperature for 30 minutes.
DNA iodinado foi ainda puri. ficado por precipitação em 3,1 ml de acetato de sódio 3M pH 5,2 em etanol a 99,5 (0,1:3) a 80°C durante 30 minutos, depois centrifugado a 15.000 rpm. Rejeitou-se o sobrenadante e dissolveu-se o precipitado em 1 ml de tampão 10 mM Tris, lmM EDTA e 10 mM NaCl pH 7,5, tornou-se a precipitar com acetato de sódio 3M pH 5,2 e etanol a 99,5% como atrás. Rejeitou-se novamente o sobrenadante e lavou-se o precipitado com etanol a 70% e secou-se.Iodinated DNA was still puri. precipitated in 3.1 ml of 3M sodium acetate pH 5.2 in ethanol at 99.5 (0.1: 3) at 80 ° C for 30 minutes, then centrifuged at 15,000 rpm. The supernatant was discarded and the precipitate was dissolved in 1 ml of 10 mM Tris buffer, 1 mM EDTA and 10 mM NaCl pH 7.5, precipitated again with 3M sodium acetate pH 5.2 and ethanol at 99, 5% as above. The supernatant was discarded again and the precipitate was washed with 70% ethanol and dried.
O DNA iodinado foi ressuspenso em solução aquosa de borato de sódio 50 mM, 15 mM EDTA e 0 ,01% de azida de sódio de modo a ter-se uma concentração final de 0,1 pli/ml. A solução resultante foi usada como traçadora com uma actividade específica de 0,3 uli/pmole.The iodinated DNA was resuspended in 50 mM aqueous sodium borate solution, 15 mM EDTA and 0.01% sodium azide in order to have a final concentration of 0.1 pL / ml. The resulting solution was used as a tracer with a specific activity of 0.3 µl / pmole.
Exemplo 3Example 3
Processo para testar anticorpos circulantes anti-DNAProcess for testing circulating anti-DNA antibodies
Tendo preparado uma marca bemHaving prepared a well
125 definida de I-ds DNA com determinados kpb e actividade específica, capaz de se ligar especificamente a imunoglobulinas circulantes anti-DNA possuíamos a base de um método para medição de títulos de anticorpos anti-DNA em amostras de soro.125 defined I-ds DNA with specific kpb and specific activity, capable of specifically binding to circulating anti-DNA immunoglobulins, we had the basis of a method for measuring anti-DNA antibody titers in serum samples.
método de radioensaio que se segue é a execução preferida do presente invento mas não é no entanto, restrictivo:The following radioassay method is the preferred embodiment of the present invention but is not, however, restrictive:
Prepararam-se calibradores anti-DNA a partir de um soro de um paciente que sofria de SLE e diluiram-se seriadamente em soro humano sem anticorpos anti-DNA. Os calibradores foram referenciados relativamente a um outro radioensaio produzido pela Amersham Corporation, Amersham England. Os calibradores foram expressos arbitrariamente em unidades/ml (u/ml).Anti-DNA calibrators were prepared from a serum from a patient suffering from SLE and diluted seriously in human serum without anti-DNA antibodies. The calibrators were referenced to another radioassay produced by the Amersham Corporation, Amersham England. The calibrators were expressed arbitrarily in units / ml (u / ml).
125125
O dsDNA marcado com I foi diluido como se mostrou atrás para dar 75000 cpm por 200 yl de volume.The I-labeled dsDNA was diluted as shown above to give 75000 cpm per 200 æl volume.
ul de cada calibrador e a amostra foram pipetados para tubos de ensaio de 12 x 75 mm aos quais se adicionou 200 ul de I-ds DNA. Após incubação durante 2 horas a 37°C, adicionou-se 1,0 ml de solução a 50% de sulfato de amónio aquoso para separar o traçador ligado à imunoglobulina do traçador livre não ligado. Após centrifugação a 2000 x g durante 15 minutos a fracção ligada foi separada da fracção livre por decantação dos tubos. Con-12tou-se o precipitado e leram-se as concentrações das amostras dos pacientes, relativamente à curva de calibração onde as contagens ligadas presentes na amostra do paciente são directamente proporcionais às unidades de actividade dos títulos anti-DNA tul of each calibrator and the sample were pipetted into 12 x 75 mm test tubes to which 200 ul of I-ds DNA was added. After incubation for 2 hours at 37 ° C, 1.0 ml of 50% aqueous ammonium sulfate solution was added to separate the immunoglobulin-bound tracer from the unbound free tracer. After centrifugation at 2000 x g for 15 minutes, the bound fraction was separated from the free fraction by decanting the tubes. The precipitate was collected and the concentrations of the patient samples were read, relative to the calibration curve where the linked counts present in the patient sample are directly proportional to the units of activity of the anti-DNA t
Exemplo 4Example 4
Efeito de Csq do complementoComplement Csq effect
Testaram-se dez amostras nor- ! mais pelo método deste invento antes e depois do tratamento pelo calor a 56°C durante 30 minutos para inactivar o complemento, Csq, usando dois traçadores. O traçador 1 foi o 125Ten normal samples were tested! more by the method of this invention before and after heat treatment at 56 ° C for 30 minutes to inactivate the complement, Csq, using two tracers. Tracer 1 was 125
I-ds DNA do Exemplo 2, supra, e é a execução preferidaI-ds DNA of Example 2, supra, and is the preferred execution
125 í deste invento. O traçador 2 foi um I-ss DNA preparado como se segue: o ds DNA recombinante (1,1 a 1,2 kpb) concentrado do Exemplo 1, supra, foi aquecido a 95°C durante um minuto, arrefecido em gelo e deixado reagir à temperatura 125 ambiente antes da iodinaçao, com I como descrito no Exempio 2, supra. O DNA resultante de cadeia simples e iodi125 nado ( I-ss DNA) foi então diluido no tampão traçador e usado no ensaio.125 of this invention. Tracer 2 was an I-ss DNA prepared as follows: the concentrated recombinant DNA (1.1 to 1.2 kbp) of Example 1, supra, was heated to 95 ° C for one minute, cooled on ice and left react at room temperature before iodination, with I as described in Example 2, supra. The resulting single-stranded, iodinated DNA (I-ss DNA) was then diluted in the tracer buffer and used in the assay.
O resultado desta experiência está resumido abaixo:The result of this experiment is summarized below:
Traçador:Tracer:
1. 1251-dsDNA1. 1251-dsDNA
2. 1251-ssDNA2. 1251-ssDNA
Os resultados mostram claramente que o traçador de DNA de cadeia dupla, traçador 1, 125The results clearly show that the double-stranded DNA tracer, tracer 1, 125
I-dsDNA,não é afectado pela presença do complemento no soro de pacientes. Por outro lado, o traçador DNA de cadeiaI-dsDNA, is not affected by the presence of complement in the serum of patients. On the other hand, the chain DNA tracer
125 simples, traçador 2, I-ssDNA é bastante afectado.125 simple, tracer 2, I-ssDNA is quite affected.
Todos os g/individuos normais testados tinham resultados anti-DNA abaixo de 6U/ml, conforme medido pelo método deste invento, enquanto todos, excepto um, os 28 pacientes com SLE activo (96,4%) tinham resultados de 20 U/ml ou superiores. O paciente excepçao com SLE activo tinha um nível anti-DNA de 5,7 U/ml. Em adição, 30 dos 48 pacientes com SLE inactivo (62,5%) tinham resultados acima de 6U/ml.All g / normal individuals tested had anti-DNA results below 6U / ml, as measured by the method of this invention, while all but one, the 28 patients with active SLE (96.4%) had results of 20 U / ml or higher. The exception patient with active SLE had an anti-DNA level of 5.7 U / ml. In addition, 30 of the 48 patients with inactive SLE (62.5%) had results above 6U / ml.
Dos 46 pacientes com perturbações do tecido conjuntivo que não SLE, apenas dois (4,4%) tinham níveis anti-DNA acima de 6U/ml.Of the 46 patients with connective tissue disorders other than SLE, only two (4.4%) had anti-DNA levels above 6U / ml.
Um nível de decisão estabeleci do aproximadamente no limite superior do normal tornaria assim máxima a sensibilidade para SLE activo nesta população minimizando ao mesmo tempo o número de resultados positivos em pacientes com perturbações diferentes de SLE.A decision level established at approximately the upper limit of normal would thus make sensitivity to active SLE in this population maximum while minimizing the number of positive results in patients with disorders other than SLE.
Tendo descrito totalmente o invento pretende-se que ele seja limitado apenas pelo âmbito legal das reivindicações apensas.Having fully described the invention, it is intended to be limited only by the legal scope of the appended claims.
Claims (5)
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1997087 | 1987-01-30 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| PT86643A PT86643A (en) | 1988-02-01 |
| PT86643B true PT86643B (en) | 1992-01-31 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PT86643A PT86643B (en) | 1987-01-30 | 1988-01-28 | METHOD OF MEASURING ANTI-DNA ANTIBODIES |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0276984A3 (en) |
| JP (1) | JPH0192660A (en) |
| KR (1) | KR880009129A (en) |
| CN (1) | CN88100541A (en) |
| AU (1) | AU1070588A (en) |
| PT (1) | PT86643B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03167473A (en) * | 1989-11-08 | 1991-07-19 | Nippon Dpc Corp | Method and kit for measuring anti-dna antibody coupling activity |
| US5681700A (en) * | 1994-05-25 | 1997-10-28 | Oklahoma Medical Research Foundation | Assay for pathogenicity of anti-DNA antibodies |
| US6280944B1 (en) | 1994-05-25 | 2001-08-28 | Oklahoma Medical Research Foundation | Assay for pathogenicity of anti-DNA antibodies |
| US5700641A (en) * | 1995-03-01 | 1997-12-23 | Salonen; Eeva-Marjatta | Diagnostic method, test kit, drug and therapeutic treatment for autoimmune diseases |
| FI100556B (en) * | 1995-03-01 | 1997-12-31 | Biohit Oyj | Procedure for Detecting Anti-DNA Antibodies, Diagnostic Procedure, Reagent Packaging and Autoimmune Disease Drugs |
| JPH08233813A (en) * | 1995-12-13 | 1996-09-13 | Nippon Dpc Corp | Method for measuring anti-double-stranded DNA antibody titer in human body fluid |
| KR20170072365A (en) | 2007-12-27 | 2017-06-26 | 백스터 인터내셔널 인코포레이티드 | Method and compositions for specifically detecting physiologically acceptable polymer molecules |
| KR102401369B1 (en) * | 2020-06-02 | 2022-05-24 | 경북대학교 산학협력단 | Titration method in anti-nuclear antibody testing for the diagnosis of autoimmune diseases |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5742632A (en) * | 1980-08-29 | 1982-03-10 | Takeshi Sasaki | Double-chain dna bonded to d-glutamic acid-d-lysine copolymer, its preparation and medicine containing the same |
| JPS5856694A (en) * | 1981-09-25 | 1983-04-04 | Mitsui Pharmaceut Inc | Reagent for assay of antinuclear antibody |
| GB8306426D0 (en) * | 1983-03-09 | 1983-04-13 | Malcolm A D B | Detecting polynucleotide sequence |
| EP0135051B1 (en) * | 1983-07-25 | 1989-01-25 | Whittaker Corporation | Immunoassay for the determination of antibodies against native dna |
| US4623627A (en) * | 1983-08-19 | 1986-11-18 | Cetus Corporation | Monoclonal antibody having specificity for the double-stranded conformation of native DNA and diagnostic methods using same |
| FR2558172B1 (en) * | 1984-01-16 | 1986-06-13 | Inst Nat Sante Rech Med | PROBE CONTAINING MODIFIED NUCLEIC ACID AND RECOGNITION BY SPECIFIC ANTIBODIES AND USE OF SUCH PROBE AND THESE SPECIFIC ANTIBODIES TO DETECT AND CHARACTERIZE A HOMOLOGATED DNA SEQUENCE |
| JPS60253869A (en) * | 1984-05-30 | 1985-12-14 | Mitsui Pharmaceut Inc | Reagent for mesuring anti-artificial nucleus antigen antibody |
| CA1272443A (en) * | 1985-02-22 | 1990-08-07 | Nanibhushan Dattagupta | Solution-phase dual hybridization assay for detecting polynucleotide sequences |
-
1988
- 1988-01-22 AU AU10705/88A patent/AU1070588A/en not_active Abandoned
- 1988-01-26 EP EP88300640A patent/EP0276984A3/en not_active Withdrawn
- 1988-01-28 JP JP8818481A patent/JPH0192660A/en active Granted
- 1988-01-28 PT PT86643A patent/PT86643B/en not_active IP Right Cessation
- 1988-01-29 KR KR1019880000752A patent/KR880009129A/en not_active Withdrawn
- 1988-01-30 CN CN198888100541A patent/CN88100541A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| EP0276984A2 (en) | 1988-08-03 |
| JPH0562301B2 (en) | 1993-09-08 |
| JPH0192660A (en) | 1989-04-11 |
| AU1070588A (en) | 1988-08-04 |
| CN88100541A (en) | 1988-09-14 |
| PT86643A (en) | 1988-02-01 |
| KR880009129A (en) | 1988-09-14 |
| EP0276984A3 (en) | 1989-05-03 |
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